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A Cholinergic-Sympathetic Pathway Primes Immunity in Hypertension and Mediates Brain-To-Spleen Communication

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A Cholinergic-Sympathetic Pathway Primes Immunity in Hypertension and Mediates Brain-To-Spleen Communication

Daniela Carnevale et al. Nat Commun.

Abstract

The crucial role of the immune system in hypertension is now widely recognized. We previously reported that hypertensive challenges couple the nervous drive with immune system activation, but the physiological and molecular mechanisms of this connection are unknown. Here, we show that hypertensive challenges activate splenic sympathetic nerve discharge to prime immune response. More specifically, a vagus-splenic nerve drive, mediated by nicotinic cholinergic receptors, links the brain and spleen. The sympathetic discharge induced by hypertensive stimuli was absent in both coeliac vagotomized mice and in mice lacking α7nAChR, a receptor typically expressed by peripheral ganglionic neurons. This cholinergic-sympathetic pathway is necessary for T cell activation and egression on hypertensive challenges. In addition, we show that selectively thermoablating the splenic nerve prevents T cell egression and protects against hypertension. This novel experimental procedure for selective splenic denervation suggests new clinical strategies for resistant hypertension.

Figures

Figure 1
Figure 1. AngII activates discharge of the splenic nerve.
(a,b) Representative raw signals of SSNA in a time bin of 10 min in (a) show a significantly increased nerve discharge in AngII-infused mice as compared with vehicle. Raw signal of residual SSNA during postmortem was used to identify the signal threshold level for each recording. In (b) it is represented an enlarged view of 10 s of SSNA raw signal from the red window in a. (c) Blood pressure (BP) monitoring during SSNA recording excluded hemodynamic effects. (d) Firing frequency, represented by the mean number of spikes in a time bin, was significantly higher in AngII-infused mice as compared with vehicle (vehicle nmice=8 and AngII nmice=10; independent samples Student's t-test, t(16)=−4.697, ***P<0.001). (e) Analysis of mean amplitude gain of spikes showed that AngII-infused mice had also increased SSNA in the pattern of burst amplitude as compared with vehicle (vehicle nmice=8 and AngII nmice=10; independent samples Student's t-test, t(9)=−3.120, **P<0.01).
Figure 2
Figure 2. Activation of splenic nerve activity is a common response to hypertensive stimuli.
(ac) DOCA-salt hypertensive challenge significantly increased splenic nerve discharge, in a similar way to that observed in AngII-infused mice, as shown by the representative raw signals of SSNA in a time bin of 10 min (a) and a particular of 10 s from the red window in b. Residual SSNA during postmortem (lower panel in a) and BP monitoring (c) are shown as well. (d) DOCA-salt induced a significant increase in firing frequency, expressed as the mean number of spikes in a time bin, as compared with mice receiving placebo (Placebo nmice=6 and DOCA-salt nmice=7; independent samples Student's t-test, t(11)=−9.654, ***P<0.001). (e) DOCA-salt-treated mice display a significant higher mean amplitude gain of spikes, as compared with mice receiving placebo alone (Placebo nmice=6 and DOCA-salt nmice=7; independent samples Student's t-test, t(11)=−5.977, ***P<0.001).
Figure 3
Figure 3. Efferent vagus nerve resection blocks the SSNA induced by hypertensive stimuli.
(a) Mice infused with AngII were subjected to cervical vagotomy (VagX) while recording SSNA. As shown by the representative raw signals of SSNA, cervical VagX completely abolishes splenic nerve activity. (b) Analysis of firing frequency confirmed the inhibitory effect of cervical VagX on AngII-induced SSNA (nmice=5; paired samples Student's t-test, t(4)=8.524, ***P<0.001). (c) Conversely, no effect of cervical VagX on the mean amplitude gain of spikes was observed, suggesting that this pattern of SSNA was regulated by pathways different from the vagus nerve (nmice=5; paired samples Student's t-test, t(4)=−2.764, P=0.051). (d) To verify whether the effect was due to the afferent or efferent branch of the vagus nerve, a further group of AngII-infused mice underwent coeliac vagotomy (VagX) while recording SSNA. Even in this case, splenic nerve activity was completely inhibited by coeliac VagX. (e) Analysis of firing frequency confirmed the inhibitory effect of coeliac VagX on AngII-induced SSNA (nmice=5; paired samples Student's t-test, t(4)=11.059, ***P<0.001). (f) Yet, no effect of coeliac VagX on the mean amplitude gain of spikes was observed, further supporting that this pattern of SSNA was regulated by pathways different from the vagus nerve (nmice=5; paired samples Student's t-test, t(4)=−1.518, P=0.204).
Figure 4
Figure 4. Coeliac vagotomy protects from AngII-induced blood pressure increase and T cell egression.
(a,b) Mice with left coeliac VagX did not become hypertensive in response to chronic AngII infusion (nmice=5 for each group; two-way ANOVA for repeated measures; (a) systolic blood pressure SBP, F(interaction)=3.321, ***P<0.001; (b) Diastolic blood pressure DBP, F(interaction)=2.284, ***P<0.001). (c) Left coeliac VagX was also effective in inhibiting the T cell egression induced by AngII, as evidenced by the area of CD3+ cells (magenta), representing the white pulp and delimited by B220+ cells (green) delineating the red pulp (scale bar, 200 μm). (d) Graph showing the relative quantitative analysis (nmice=5 for each group; two-way ANOVA, F(interaction)=20.160, **P<0.01).
Figure 5
Figure 5. α7nAChR is necessary to the activation of SSNA on hypertensive challenges.
(a–c) The representative raw signals of SSNA in a time bin (a) and in an enlarged particular of 10 s (b) show that α7nAChR KO mice have a reduced response to AngII infusion, as compared with WT controls. Residual SSNA during postmortem (lower panel in a) and blood pressure monitoring (c) are shown as well. (d) Analysis of firing frequency confirmed the reduced activity of α7nAChR KO mice on AngII infusion (WT and α7nAChR KO nmice=7; independent samples Student's t-test, t(12)=3.493, **P<0.01). (e) Accordingly to what observed in VagX, the mean amplitude gain of spikes induced by AngII was unaffected in α7nAChR KO, showing levels comparable to that of WT mice (WT and α7nAChR KO nmice=7; independent samples Student's t-test, t(12)=0.572, P=0.578).
Figure 6
Figure 6. α7nAChR KO mice are protected from AngII-induced hypertension and T cell egression.
(a,b) α7nAChR KO mice were protected from hypertension induced by chronic AngII infusion (nmice=8 for each group; two-way ANOVA for repeated measures; (a) SBP, F(interaction)=5.331, ***P<0.001; (b) DBP, F(interaction)=5.087, ***P<0.001). (c) Ablation of α7nAChR was effective in blocking T cell egression on AngII, similarly to vagotomized mice, as evidenced by the area of white pulp, labelled by CD3+ cells (magenta), delimited by B220+ cells (green) of the red pulp (scale bar, 200 μm). (d) Graph showing the relative quantitative analysis (nmice=7 WT vehicle; 8 WT AngII; 6 α7nAChR KO Vehicle; 6 α7nAChR KO AngII; two-way ANOVA, F(interaction)=8.939, *P<0.05 and ***P<0.001).
Figure 7
Figure 7. Establishment of a procedure for selective splenic denervation.
(a) Example of surgical procedure for selective SDN, showing the splenic artery before (upper panel) and after (lower panel) denervation in a representative animal. (b) Ultrasound Doppler imaging showing a normal pulse wave of the splenic artery in splenic denervated mice, comparable to that of sham. Colour Doppler also indicate a normal perfusion of the spleen after denervation. (c) Anatomical transversal reconstruction of microCT angiography evidencing of correct uptake of the contrast agent in the spleen. (d) 3D reconstruction demonstrating integrity of splenic artery after denervation and consequent normal perfusion of the spleen.
Figure 8
Figure 8. Selective splenic denervation blocks sympathetic nervous system in the spleen.
(a) Coeliac ganglion neurons, labelled with a retrograde neurotracer injected in the splenic parenchyma, are significantly reduced in SDN mice as compared with sham controls. (nmice=6 for each group; scale bar, 200 μm). (b) Significant reduction of tyrosine hydroxylase immunofluorescence in splenic artery of two representative SDN as compared with Sham mice (nmice=6 for each group; scale bar, 50 μm). (c) Noradrenaline levels were significantly reduced in the spleen of SDN mice as compared with Sham mice (Sham and SDN groups nmice=8; independent samples Student's t-test, t(14)=10.606, ***P<0.001). (d) Analysis of noradrenaline content in the ipsilateral kidney was unaltered in SDN mice as compared with Sham, revealing no off-target effect of the procedure (Sham and SDN groups nmice=8; independent samples Student's t-test, t(14)=0.853, P=0.408).
Figure 9
Figure 9. Splenic denervation protects from hypertension and T cell egression from spleen.
(a,b) Mice with splenic denervation were protected from AngII-induced hypertension, as compared with Sham mice and SDN mice with vehicle alone (Sham-vehicle, Sham-AngII and SDN-vehicle nmice=10; SDN-AngII nmice=11; two-way ANOVA for repeated measures; (a) SBP, F(interaction)=6.723, ***P<0.001; (b) DBP, F(interaction)=3.070, ***P<0.001). (c) Mice with splenic denervation are protected from the T cell egression induced by AngII, as evidenced by the area of CD3+ cells (magenta), recognizing white pulp and delimited by B220+ cells (green) delineating red pulp (nmice=5 for each group; scale bar, 200 μm). (d) Graph showing the relative quantitative analysis (nmice=5 for each group; two-way ANOVA, F(interaction)=46.384, ***P<0.001). (e) AngII fails to induce co-stimulation of T cells in mice with splenic denervation, as evidenced by reduced CD86+ cells (green) expression in the marginal zone of the spleen, labelled by CD169+ cells (magenta) (nmice=5 for each group; scale bar, 50 μm).
Figure 10
Figure 10. Splenic denervation protects from the T cells infiltration in target organs of hypertension.
(a,b) Flow cytometry analysis shows that SDN mice were protected from the AngII-induced increase of total CD8+ and CD4+ T cells (upper panels) in aorta (a) and (b) kidneys. SDN mice displayed also a reduced amount of cells positive for homing (CD44) and activation (CD69) antigens (middle and lower panels, respectively) (nmice=9 for each group). Significance of two-way ANOVA was as follow for aortas: CD8 F(interaction)=7.922, CD8-CD44 F(interaction)=5.071, CD8-CD69 F(interaction)=7.344, CD4 F(interaction)=9.122, CD4-CD44 F(interaction)=7.867, CD4-CD69 F(interaction)=8.432. Significance of Two-way ANOVA was as follow for kidneys: CD8 F(interaction)=13.88, CD8-CD44 F(interaction)=5.382, CD8-CD69 F(interaction)=7.202, CD4 F(interaction)=6.248, CD4-CD44 F(interaction)=3.810, CD4-CD69 F(interaction)=10.89. *P<0.05, **P<0.01 and ***P<0.001. (c) Representative plots and gating strategies are shown for kidneys.

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